Search

The increase in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) and upregulation of calcium-sensing receptor (CaSR) and stromal interaction molecule 2 (STIM2) along with inhibition of voltage-gated K(+) (K(V)) channels in pulmonary arterial smooth muscle cells (PASMC) have been implicated in the development of pulmonary arterial hypertension; however, the precise upstream mechanisms remain elusive. Activation of CaSR, a G protein-coupled receptor (GPCR), results in Ca(2+) release from the endoplasmic/sarcoplasmic reticulum (ER/SR) and Ca(2+) influx through receptor-operated and store-operated Ca(2+) channels (SOC). Upon Ca(2+) depletion from the SR, STIM forms clusters to mediate store-operated Ca(2+) entry. Activity of K(V) channels, like KCNA5/K(V)1.5 and KCNA2/K(V)1.2, contributes to regulating membrane potential, and inhibition of K(V) channels results in membrane depolarization that increases [Ca(2+)](cyt) by opening voltage-dependent Ca(2+) channels. In this study, we show that activation of Notch by its ligand Jag-1 promotes the clustering of STIM2, and clustered STIM2 subsequently enhances the CaSR-induced Ca(2+) influx through SOC channels. Extracellular Ca(2+)-mediated activation of CaSR increases [Ca(2+)](cyt) in CASR-transfected HEK293 cells. Treatment of CASR-transfected cells with Jag-1 further enhances CaSR-mediated increase in [Ca(2+)](cyt). Moreover, CaSR-mediated increase in [Ca(2+)](cyt) was significantly augmented in cells co-transfected with CASR and STIM2. CaSR activation results in STIM2 clustering in CASR/STIM2-cotransfected cells. Notch activation also induces significant clustering of STIM2. Furthermore, activation of Notch attenuates whole cell K(+) currents in KCNA5- and KCNA2-transfected cells. Together, these results suggest that Notch activation enhances CaSR-mediated increases in [Ca(2+)](cyt) by enhancing store-operated Ca(2+) entry and inhibits KCNA5/K(V)1.5 and KCNA2/K(V)1.2, ultimately leading to voltage-activated Ca(2+) entry.

Ca(2+) signaling, particularly the mechanism via store-operated Ca(2+) entry (SOCE) and receptor-operated Ca(2+) entry (ROCE), plays a critical role in the development of acute hypoxia-induced pulmonary vasoconstriction and chronic hypoxia-induced pulmonary hypertension. This study aimed to test the hypothesis that chronic hypoxia differentially regulates the expression of proteins that mediate SOCE and ROCE [stromal interacting molecule (STIM), Orai, and canonical transient receptor potential channel TRPC6] in pulmonary (PASMC) and coronary (CASMC) artery smooth muscle cells. The resting cytosolic [Ca(2+)] ([Ca(2+)](cyt)) and the stored [Ca(2+)] in the sarcoplasmic reticulum were not different in CASMC and PASMC. Seahorse measurement showed a similar level of mitochondrial bioenergetics (basal respiration and ATP production) between CASMC and PASMC. Glycolysis was significantly higher in PASMC than in CASMC. The amplitudes of cyclopiazonic acid-induced SOCE and OAG-induced ROCE in CASMC are slightly, but significantly, greater than in PASMC. The frequency and the area under the curve of Ca(2+) oscillations induced by ATP and histamine were also larger in CASMC than in PASMC. Na(+)/Ca(2+) exchanger-mediated increases in [Ca(2+)](cyt) did not differ significantly between CASMC and PASMC. The basal protein expression levels of STIM1/2, Orai1/2, and TRPC6 were higher in CASMC than in PASMC, but hypoxia (3% O(2) for 72 h) significantly upregulated protein expression levels of STIM1/STIM2, Orai1/Orai2, and TRPC6 and increased the resting [Ca(2+)](cyt) only in PASMC, but not in CASMC. The different response of essential components of store-operated and receptor-operated Ca(2+) channels to hypoxia is a unique intrinsic property of PASMC, which is likely one of the important explanations why hypoxia causes pulmonary vasoconstriction and induces pulmonary vascular remodeling, but causes coronary vasodilation.

Two prescient 1953 publications set the stage for the elucidation of a novel endocrine system: Schatzmann’s report that cardiotonic steroids (CTSs) are all Na+ pump inhibitors, and Szent-Gyorgi’s suggestion that there is an endogenous “missing screw” in heart failure that CTSs like digoxin may replace. In 1977 I postulated that an endogenous Na+ pump inhibitor acts as a natriuretic hormone and simultaneously elevates blood pressure (BP) in salt-dependent hypertension. This hypothesis was based on the idea that excess renal salt retention promoted the secretion of a CTS-like hormone that inhibits renal Na+ pumps and salt reabsorption. The hormone also inhibits arterial Na+ pumps, elevates myocyte Na+ and promotes Na/Ca exchanger-mediated Ca2+ gain. This enhances vasoconstriction and arterial tone—the hallmark of hypertension. Here I describe how those ideas led to the discovery that the CTS-like hormone is endogenous ouabain (EO), a key factor in the pathogenesis of hypertension and heart failure. Seminal observations that underlie the still-emerging picture of the EO-Na+ pump endocrine system in the physiology and pathophysiology of multiple organ systems are summarized. Milestones include: 1) cloning the Na+ pump isoforms and physiological studies of mutated pumps in mice; 2) discovery that Na+ pumps are also EO-triggered signaling molecules; 3) demonstration that ouabain, but not digoxin, is hypertensinogenic; 4) elucidation of EO’s roles in kidney development and cardiovascular and renal physiology and pathophysiology; 5) discovery of “brain ouabain”, a component of a novel hypothalamic neuromodulatory pathway; and 6) finding that EO and its brain receptors modulate behavior and learning.

Pulmonary arterial hypertension (PAH) is a progressive disease that, if left untreated, eventually leads to right heart failure and death. Elevated pulmonary arterial pressure (PAP) in patients with PAH is mainly caused by an increase in pulmonary vascular resistance (PVR). Sustained vasoconstriction and excessive pulmonary vascular remodeling are two major causes for elevated PVR in patients with PAH. Excessive pulmonary vascular remodeling is mediated by increased proliferation of pulmonary arterial smooth muscle cells (PASMC) due to PASMC dedifferentiation from a contractile or quiescent phenotype to a proliferative or synthetic phenotype. Increased cytosolic Ca2+concentration ([Ca2+]cyt) in PASMC is a key stimulus for cell proliferation and this phenotypic transition. Voltage-dependent Ca2+entry (VDCE) and store-operated Ca2+entry (SOCE) are important mechanisms for controlling [Ca2+]cyt. Stromal interacting molecule proteins (e.g., STIM2) and Orai2 both contribute to SOCE and we have previously shown that STIM2 and Orai2, specifically, are upregulated in PASMC from patients with idiopathic PAH and from animals with experimental pulmonary hypertension in comparison to normal controls. In this study, we show that STIM2 and Orai2 are upregulated in proliferating PASMC compared with contractile phenotype of PASMC. Additionally, a switch in Ca2+regulation is observed in correlation with a phenotypic transition from contractile PASMC to proliferative PASMC. PASMC in a contractile phenotype or state have increased VDCE, while in the proliferative phenotype or state PASMC have increased SOCE. The data from this study indicate that upregulation of STIM2 and Orai2 is involved in the phenotypic transition of PASMC from a contractile state to a proliferative state; the enhanced SOCE due to upregulation of STIM2 and Orai2 plays an important role in PASMC proliferation.

H2S generated by the enzyme cystathionine-γ-lyase (CSE) has been implicated in O2 sensing by the carotid body. The objectives of the present study were to determine whether glomus cells, the primary site of hypoxic sensing in the carotid body, generate H2S in an O2-sensitive manner and whether endogenous H2S is required for O2 sensing by glomus cells. Experiments were performed on glomus cells harvested from anesthetized adult rats as well as age and sex-matched CSE+/+ and CSE−/− mice. Physiological levels of hypoxia (Po2 ∼30 mmHg) increased H2S levels in glomus cells, and dl-propargylglycine (PAG), a CSE inhibitor, prevented this response in a dose-dependent manner. Catecholamine (CA) secretion from glomus cells was monitored by carbon-fiber amperometry. Hypoxia increased CA secretion from rat and mouse glomus cells, and this response was markedly attenuated by PAG and in cells from CSE−/− mice. CA secretion evoked by 40 mM KCl, however, was unaffected by PAG or CSE deletion. Exogenous application of a H2S donor (50 μM NaHS) increased cytosolic Ca2+ concentration ([Ca2+]i) in glomus cells, with a time course and magnitude that are similar to that produced by hypoxia. [Ca2+]i responses to NaHS and hypoxia were markedly attenuated in the presence of Ca2+-free medium or cadmium chloride, a pan voltage-gated Ca2+ channel blocker, or nifedipine, an L-type Ca2+ channel inhibitor, suggesting that both hypoxia and H2S share common Ca2+-activating mechanisms. These results demonstrate that H2S generated by CSE is a physiologic mediator of the glomus cell's response to hypoxia.

ion channels regulate the selective transport of ions and are fundamental to numerous physiological processes; including, but not limited to, muscle contraction, neuron firing and cellular homeostasis. The EAG or “ ether-a-go-go ” gene encodes a family of depolarization-activated or voltage

in this issue, the American Journal of Physiology-Cell Physiology initiates the publication of a new Theme series of review articles that focus on the pathogenic role of ion channels and transporters in cancer. Ion channels, as an important family of membrane proteins that are highly expressed in

Factors contributing to the development of a fibrotic vascular scar and pulmonary vascular remodeling leading to chronic thromboembolic pulmonary hypertension (CTEPH) are still unknown. This study investigates the potential contribution of multipotent progenitor cells and myofibroblasts to the development and progression of CTEPH. Histological examination of endarterectomized tissues from patients with CTEPH identified significant neointimal formation. Morphological heterogeneity was observed in cells isolated from these tissues, including a network-like growth pattern and the formation of colony-forming unit-fibroblast-like colonies (CFU-F). Cells typically coexpressed intermediate filaments vimentin and smooth muscle α-actin. Cells were characterized by immunofluorescence and quantitated by fluorescent-activated cell sorting (FACS) for the presence of cell surface markers typical of mesenchymal progenitor cells; cells were >99% CD44+ CD73+, CD90+, CD166+; >80% CD29+; 45–99% CD105+; CD34− and CD45−. Cells were capable of adipogenic and osteogenic differentiation, determined by Oil Red O and Alizarin Red staining, respectively. Additionally, a population of Stro-1+ cells, a marker of bone marrow-derived stromal cells (4.2%), was sorted by FACS and also capable of adipogenic and osteogenic differentiation. In conclusion, this study is the first to identify a myofibroblast cell phenotype to be predominant within endarterectomized tissues, contributing extensively to the vascular lesion/clot. This cell may arise from transdifferentiation of adventitial fibroblasts or differentiation of mesenchymal progenitor cells. The unique microenvironment created by the stabilized clot is likely a factor in stimulating such cellular changes. These findings will be critical in establishing future studies in the development of novel and much needed therapeutic approaches for pulmonary hypertension.

Intestinal mucosal restitution occurs as a consequence of epithelial cell migration and reseals superficial wounds after injury. This rapid reepithelialization is mediated in part by a phospholipase C-γ1 (PLC-γ1)-induced Ca2+ signaling, but the exact mechanism underlying such signaling and its regulation remains elusive. The small GTP-binding protein Rac1 functions as a pivotal regulator of several signaling networks and plays an important role in regulating cell motility. The current study tests the hypothesis that Rac1 modulates intestinal epithelial cell migration after wounding by altering PLC-γ1-induced Ca2+ signaling. Inhibition of Rac1 activity by treatment with its inhibitor NSC-23766 or Rac1 silencing with small interfering RNA decreased store depletion-induced Ca2+ influx and suppressed cell migration during restitution, whereas ectopic overexpression of Rac1 increased Ca2+ influx and promoted cell migration. Rac1 physically interacted with PLC-γ1 and formed Rac1/PLC-γ1 complex in intestinal epithelial cells. PLC-γ1 silencing in cells overexpressing Rac1 prevented stimulation of store depletion-induced Ca2+ influx and cell migration after wounding. Polyamine depletion inhibited expression of both Rac1 and PLC-γ1, decreased Rac1/PLC-γ1 complex levels, reduced Ca2+ influx, and repressed cell migration. Overexpression of Rac1 alone failed to rescue Ca2+ influx after store depletion and cell migration in polyamine-deficient cells, because it did not alter PLC-γ1 levels. These results indicate that Rac1 promotes intestinal epithelial cell migration after wounding by increasing Ca2+ influx as a result of its interaction with PLC-γ1.

A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) is a trigger for pulmonary vasoconstriction and a stimulus for PASMC proliferation and migration. Multiple mechanisms are involved in regulating [Ca2+]cyt in human PASMC. The resting [Ca2+]cyt and Ca2+ entry are both increased in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH), which is believed to be a critical mechanism for sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in these patients. Here we report that protein expression of NCX1, an NCX family member of Na+/Ca2+ exchanger proteins is upregulated in PASMC from IPAH patients compared with PASMC from normal subjects and patients with other cardiopulmonary diseases. The Na+/Ca2+ exchanger operates in a forward (Ca2+ exit) and reverse (Ca2+ entry) mode. By activating the reverse mode of Na+/Ca2+ exchange, removal of extracellular Na+ caused a rapid increase in [Ca2+]cyt, which was significantly enhanced in IPAH PASMC compared with normal PASMC. Furthermore, passive depletion of intracellular Ca2+ stores using cyclopiazonic acid (10 μM) not only caused a rise in [Ca2+]cyt due to Ca2+ influx through store-operated Ca2+ channels but also mediated a rise in [Ca2+]cyt via the reverse mode of Na+/Ca2+ exchange. The upregulated NCX1 in IPAH PASMC led to an enhanced Ca2+ entry via the reverse mode of Na+/Ca2+ exchange, but did not accelerate Ca2+ extrusion via the forward mode of Na+/Ca2+ exchange. These observations indicate that the upregulated NCX1 and enhanced Ca2+ entry via the reverse mode of Na+/Ca2+ exchange are an additional mechanism responsible for the elevated [Ca2+]cyt in PASMC from IPAH patients.

Agonist stimulation of human pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC) with histamine showed similar spatiotemporal patterns of Ca2+release. Both sustained elevation and oscillatory patterns of changes in cytosolic Ca2+concentration ([Ca2+]cyt) were observed in the absence of extracellular Ca2+. Capacitative Ca2+entry (CCE) was induced in PASMC and PAEC by passive depletion of intracellular Ca2+stores with 10 μM cyclopiazonic acid (CPA; 15–30 min). The pyrazole derivative BTP2 inhibited CPA-activated Ca2+influx, suggesting that depletion of CPA-sensitive internal stores is sufficient to induce CCE in both PASMC and PAEC. The recourse of histamine-mediated Ca2+release was examined after exposure of cells to CPA, thapsigargin, caffeine, ryanodine, FCCP, or bafilomycin. In PASMC bathed in Ca2+-free solution, treatment with CPA almost abolished histamine-induced rises in [Ca2+]cyt. In PAEC bathed in Ca2+-free solution, however, treatment with CPA eliminated histamine-induced sustained and oscillatory rises in [Ca2+]cytbut did not affect initial transient increase in [Ca2+]cyt. Furthermore, treatment of PAEC with a combination of CPA (or thapsigargin) and caffeine (and ryanodine), FCCP, or bafilomycin did not abolish histamine-induced transient [Ca2+]cytincreases. These observations indicate that 1) depletion of CPA-sensitive stores is sufficient to cause CCE in both PASMC and PAEC; 2) induction of CCE in PAEC does not require depletion of all internal Ca2+stores; 3) the histamine-releasable internal stores in PASMC are mainly CPA-sensitive stores; 4) PAEC, in addition to a CPA-sensitive functional pool, contain other stores insensitive to CPA, thapsigargin, caffeine, ryanodine, FCCP, and bafilomycin; and 5) although the CPA-insensitive stores in PAEC may not contribute to CCE, they contribute to histamine-mediated Ca2+release.

A rise in cytosolic Ca2+concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) is an important stimulus for cell contraction, migration, and proliferation. Depletion of intracellular Ca2+stores opens store-operated Ca2+channels (SOC) and causes Ca2+entry. Transient receptor potential (TRP) cation channels that are permeable to Na+and Ca2+are believed to form functional SOC. Because sarcolemmal Na+/Ca2+exchanger has also been implicated in regulating [Ca2+]cyt, this study was designed to test the hypothesis that the Na+/Ca2+exchanger (NCX) in cultured human PASMC is functionally involved in regulating [Ca2+]cytby contributing to store depletion-mediated Ca2+entry. RT-PCR and Western blot analyses revealed mRNA and protein expression for NCX1 and NCKX3 in cultured human PASMC. Removal of extracellular Na+, which switches the Na+/Ca2+exchanger from the forward (Ca2+exit) to reverse (Ca2+entry) mode, significantly increased [Ca2+]cyt, whereas inhibition of the Na+/Ca2+exchanger with KB-R7943 (10 μM) markedly attenuated the increase in [Ca2+]cytvia the reverse mode of Na+/Ca2+exchange. Store depletion also induced a rise in [Ca2+]cytvia the reverse mode of Na+/Ca2+exchange. Removal of extracellular Na+or inhibition of the Na+/Ca2+exchanger with KB-R7943 attenuated the store depletion-mediated Ca2+entry. Furthermore, treatment of human PASMC with KB-R7943 also inhibited cell proliferation in the presence of serum and growth factors. These results suggest that NCX is functionally expressed in cultured human PASMC, that Ca2+entry via the reverse mode of Na+/Ca2+exchange contributes to store depletion-mediated increase in [Ca2+]cyt, and that blockade of the Na+/Ca2+exchanger in its reverse mode may serve as a potential therapeutic approach for treatment of pulmonary hypertension.

Extracellular ATP and intracellular cyclic AMP response element-binding protein (CREB, a transcription factor) promote cell proliferation in many cell types. The canonical transient receptor potential (TRPC) channels, which putatively participate in forming store- and receptor-operated Ca2+channels, have been implicated in the pulmonary vascular remodeling processes. A link between extracellular ATP, CREB activation, and TRPC4 channel expression and activity has not been shown in human pulmonary artery smooth muscle cells (PASMC). Long-term (24–48 h) treatment of human PASMC with a low dose (100 μM) of ATP, which did not trigger a transient rise in free cytosolic Ca2+concentration ([Ca2+]i) when applied acutely to the cells, caused marked increases in CREB phosphorylation and TRPC4 protein expression. The time course indicated that the ATP-mediated CREB phosphorylation preceded TRPC4 upregulation, whereas transfection of a nonphosphorylatable CREB mutant abolished ATP-mediated TRPC4 expression. Furthermore, treatment of human PASMC with ATP also enhanced the amplitude of capacitative Ca2+entry (CCE) induced by passive store depletion, whereas the small interfering RNA specifically targeting TRPC4 attenuated ATP-mediated increases in TRPC4 expression and CCE amplitude and inhibited ATP-induced PASMC proliferation. These data suggest that low-dose ATP exerts part of its mitogenic effect in human PASMC via CREB-mediated upregulation of TRPC4 channel expression and activity and the subsequent increase in CCE and [Ca2+]i.

Capacitative Ca2+entry (CCE) through store-operated Ca2+(SOC) channels plays an important role in returning Ca2+to the sarcoplasmic reticulum (SR) and regulating cytosolic free Ca2+concentration ([Ca2+]cyt). A rise in [Ca2+]cytand sufficient Ca2+in the SR are required for pulmonary artery smooth muscle cell (PASMC) proliferation. We tested the hypothesis that platelet-derived growth factor (PDGF)-mediated PASMC growth involves upregulation of c-Jun and TRPC6, a transient receptor potential cation channel. In rat PASMC, PDGF (10 ng/ml for 0.5–48 h) phosphorylated signal transducer and activator of transcription (STAT3), increased mRNA and protein levels of c-Jun, and stimulated cell proliferation. PDGF treatment also upregulated TRPC6 expression and augmented CCE, elicited by passive depletion of Ca2+from the SR using cyclopiazonic acid. Furthermore, overexpression of c-Jun stimulated TRPC6 expression and CCE amplitude in PASMC. Downregulation of TRPC6 using an antisense oligonucleotide specifically for human TRPC6 decreased CCE and inhibited PDGF-mediated PASMC proliferation. These results suggest that PDGF-mediated PASMC proliferation is associated with c-Jun/STAT3-induced upregulation of TRPC6 expression. The resultant increase in CCE raises [Ca2+]cyt, facilitates return of Ca2+to the SR, and enhances PASMC growth.

Cell shrinkage is an early prerequisite for apoptosis. The apoptotic volume decrease is due primarily to loss of cytoplasmic ions. Increased outward K+ currents have indeed been implicated in the early stage of apoptosis in many cell types. We found that cytoplasmic dialysis of cytochrome c(cyt- c), a mitochondria-dependent apoptotic inducer, increases K+ currents before inducing nuclear condensation and breakage in pulmonary vascular smooth muscle cells. The cyt- c-mediated increase in K+ currents took place rapidly and was not affected by treatment with a specific inhibitor of caspase-9. Cytoplasmic dialysis of recombinant (active) caspase-9 negligibly affected the K+ currents. Furthermore, treatment of the cells with staurosporine (ST), an apoptosis inducer that mediates translocation of cyt- c from mitochondria to the cytosol, also increased K+ currents, caused cell shrinkage, and induced apoptosis (determined by apoptotic nuclear morphology and TdT-UTP nick end labeling assay). The staurosporine-induced increase in K+ currents concurred to the volume decrease but preceded the activation of apoptosis (nuclear condensation and breakage). These results suggest that the cyt- c-induced activation of K+ channels and the resultant K+ loss play an important role in initiating the apoptotic volume decrease when cells undergo apoptosis.

Early mucosal restitution occurs by epithelial cell migration to reseal superficial wounds after injury. Differentiated intestinal epithelial cells induced by forced expression of the Cdx2 gene migrate over the wounded edge much faster than undifferentiated parental cells in an in vitro model. This study determined whether these differentiated intestinal epithelial cells exhibit increased migration by altering voltage-gated K+ (Kv) channel expression and cytosolic free Ca2+ concentration ([Ca2+]cyt). Stable Cdx2-transfected IEC-6 cells (IEC-Cdx2L1) with highly differentiated phenotype expressed higher basal levels of Kv1.1 and Kv1.5 mRNAs and proteins than parental IEC-6 cells. Neither IEC-Cdx2L1 cells nor parental IEC-6 cells expressed voltage-dependent Ca2+ channels. The increased expression of Kv channels in differentiated IEC-Cdx2L1 cells was associated with an increase in whole cell K+ currents, membrane hyperpolarization, and a rise in [Ca2+]cyt. The migration rates in differentiated IEC-Cdx2L1 cells were about four times those of parental IEC-6 cells. Inhibition of Kv channel expression by polyamine depletion decreased [Ca2+]cyt, reduced myosin stress fibers, and inhibited cell migration. Elevation of [Ca2+]cyt by ionomycin promoted myosin II stress fiber formation and increased cell migration. These results suggest that increased migration of differentiated intestinal epithelial cells is mediated, at least partially, by increasing Kv channel activity and Ca2+ influx during restitution.

Cell shrinkage is an incipient hallmark of apoptosis in a variety of cell types. The apoptotic volume decrease has been demonstrated to attribute, in part, to K+efflux; blockade of plasmalemmal K+channels inhibits the apoptotic volume decrease and attenuates apoptosis. Using combined approaches of gene transfection, single-cell PCR, patch clamp, and fluorescence microscopy, we examined whether overexpression of Bcl-2, an anti-apoptotic oncoprotein, inhibits apoptosis in pulmonary artery smooth muscle cells (PASMC) by diminishing the activity of voltage-gated K+(Kv) channels. A human bcl-2gene was infected into primary cultured rat PASMC using an adenoviral vector. Overexpression of Bcl-2 significantly decreased the amplitude and current density of Kv currents ( IKv). In contrast, the apoptosis inducer staurosporine (ST) enhanced IKv. In bcl-2-infected cells, however, the ST-induced increase in IKvwas completely abolished, and the ST-induced apoptosis was significantly inhibited compared with cells infected with an empty adenovirus (− bcl-2). Blockade of Kv channels in control cells (− bcl-2) by 4-aminopyridine also inhibited the ST-induced increase in IKvand apoptosis. Furthermore, overexpression of Bcl-2 accelerated the inactivation of IKvand downregulated the mRNA expression of the pore-forming Kv channel α-subunits (Kv1.1, Kv1.5, and Kv2.1). These results suggest that inhibition of Kv channel activity may serve as an additional mechanism involved in the Bcl-2-mediated anti-apoptotic effect on vascular smooth muscle cells.

Intracellular K+plays an important role in controlling the cytoplasmic ion homeostasis for maintaining cell volume and inhibiting apoptotic enzymes in the cytosol and nucleus. Cytoplasmic K+concentration is mainly regulated by K+uptake via Na+-K+-ATPase and K+efflux through K+channels in the plasma membrane. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore that dissipates the H+gradient across the inner membrane of mitochondria, induces apoptosis in many cell types. In rat and human pulmonary artery smooth muscle cells (PASMC), FCCP opened the large-conductance, voltage- and Ca2+-sensitive K+(maxi-K) channels, increased K+currents through maxi-K channels [ IK(Ca)], and induced apoptosis. Tetraethylammonia (1 mM) and iberiotoxin (100 nM) decreased IK(Ca)by blocking the sarcolemmal maxi-K channels and inhibited the FCCP-induced apoptosis in PASMC cultured in media containing serum and growth factors. Furthermore, inhibition of K+efflux by raising extracellular K+concentration from 5 to 40 mM also attenuated PASMC apoptosis induced by FCCP and the K+ionophore valinomycin. These results suggest that FCCP-mediated apoptosis in PASMC is partially due to an increase of maxi-K channel activity. The resultant K+loss through opened maxi-K channels may serve as a trigger for cell shrinkage and caspase activation, which are major characteristics of apoptosis in pulmonary vascular smooth muscle cells.